Our long-term goal is to define connections between replication stress and epigenetic states and processes in human cells. The importance of this research domain to human health is underscored by the notion that epigenetic changes are expected to be more easily reversible than a genetic mutation or deletion, can thus they can hold greater potential for therapeutic manipulation. Replication stress is a state of genomic replication characterized by abnormal density, distribution, and stability of replication forks. Replication stress can be triggered by chemotherapy. As a source of genomic instability it is also implicated in early steps of carcinogenesis. The important questions that emerge from recent studies are whether epigenetic factors modulate cellular resistance to replication stress, and conversely, whether replication stress can challenge or compromise epigenetic inheritance thus opening another avenue to cellular degeneration or transformation. We will address these questions by dissecting a specific problem: chromatin modification and remodeling around moving and stalling replication forks and its roles in the context of a deficiency in the RECQ helicase WRN, mutated in the Werner syndrome of premature aging. On the one hand, recent work now implicates WRN in chromatin maintenance and epigenetic stability. On the other hand, we have previously shown that WRN absence compromises cellular resistance to replication stress and, more recently, that histone deacetylases HDAC1 and 2 cooperate with WRN in counteracting replication fork inactivation during the replication stress caused by nucleotide pool depletion. With the aim of a greater understanding of the connections between altered replication, altered chromatin, and the cellular biology of WRN deficiency, we will determine the mechanism of cooperation between WRN and HDACs in the context of nascent chromatin maturation and epigenetic changes occurring during and after replication stress in normal and WRN-deficient cells, and their effect on cell survival, proliferation, and lifespan. Our approach integrates standard cellular biology assays with high-resolution, functional analyses of genomic replication in vivo at DNA and protein levels (respectively, microfluidic-assisted replication track analysis or ma-RTA, and immunoprecipitation of nascent DNA, or iPOND). We will use RNAi and CRISPR/Cas9 manipulation to inactivate expression of the genes of interest. We will also use both targeted and unbiased approaches to identify the factors and processes involved in the functional interaction between WRN and HDAC1 and 2. We will query for specific protein candidates' involvement as well as perform mass spectrometric analyses of proteins associated with stalled replication forks in WRN or HDAC1,2-deficient and normal cells. We will also use a previously validated pipeline to perform a siRNA screen for epigenetic modifiers of HDAC, WRN- dependent replication stress phenotypes.